Functional porous substrates for attaching biomolecules

ABSTRACT

A substrate comprising a microporous microstructure, an interlayer over at least a portion of the microstructure and a functional layer attached to the interlayer, the functional layer having functional sites with a density of at least 50 nanomoles/cm2.

RELATED APPLICATION

The present application is a divisional application of allowed U.S.patent application Ser. No. 11/407,882 filed Apr. 19, 2006 now U.S. Pat.No. 7,923,054.

FIELD OF THE INVENTION

This invention relates to functional porous substrates and, moreparticularly, to such substrates used in a microarray application fordetection of biomolecules.

BACKGROUND OF THE INVENTION

Owing to their high throughput screening capability, microarrays havebecome an essential tool for the healthcare and pharmaceuticalindustries where researchers are working to diagnose disease or discovernew drugs. Moreover, agriculture and homeland defense firms areutilizing microarrays to uncover information regarding the presence ofharmful pathogenic bacteria. Such simultaneous screening is possible byprinting many microscopic spots, typically 10-250 μm in size, ofbiological molecules (i.e., biomolecules such as nucleic acid fragments,antibodies, peptides, proteins, pathogens, cells and the like) as probesonto the same substrate to form a microarray. A high density microarraydeveloped for research purposes typically comprises between 1000 to50000 probe spots arranged in a predetermined regular pattern on asubstrate, thus leading to a spot density of about 50 spots/cm² to 2500spots/cm². The dimension of the substrate can vary, but generally thesubstrate is the size of a 1 inch by 3 inch microscope slide. It iscritical that the substrate surface be reactive and capable of bindingprobe biomolecules of known sequence. In use, the microarray ishybridized with target bio-molecules of unknown sequence in order tosimultaneously detect the response of the target with the differentprobes spotted on the array surface. Typically, targets are labeled withfluorescent dyes and fluorescence based detection techniques are mostcommonly used to quantify the response of the target biomolecule to theprobes following hybridization. The composite quantitative response ofthe target to all the probes spotted on the microarray substrate is thedata resulting from the microarray experiment.

Microarray experiments can be employed to detect the expression levelsof various genes or proteins for a given organism (i.e. human, mouse,plant, bacteria, etc). Highly expressed genes or proteins are mucheasier to detect because their concentration in a given sample is oftenthe greatest. However, when expression levels are low or samples arescarce, sensitive and reliable detection technology becomes critical.This type of detection technology is increasingly important for studyingprotein-protein interactions or protein biochemical activity since theconcentration of proteins can not be amplified via enzymatic reactionssuch as the polymerase chain reaction.

As a result, within the microarray industry, there is an overriding needfor confident detection of low abundant protein and/or nucleic acids.When attempting to accurately measure or detect such low levels in amicroarray experiment, it is imperative that researchers employ systemcomponents that maximize sensitivity and overall signal to noise ratio.A number of approaches can be employed to impact sensitivity and signalto noise ratio and three of the common ones are as follows: (1)improvements in the sensitivity or detection limits of scanning devices,(2) increased amplification of the fluorescent signal via labelingmethods, and (3) the employment of a highly sensitive substrate. Thepresent invention focuses on enhancing signal to noise ratio through theemployment of a highly sensitive microarray substrate.

An increase in signal strength can be achieved by increasing the numberof binding sites per unit area (functional site density), whichultimately impacts the retention of immobilized bio-molecular probes andthe emission of an increased signal when hybridized with fluorescentlylabeled target molecules. Signal clarity can also be enhanced through areduction in the inherent auto-fluorescence of the materials and/orsystem used for detection. These approaches will ultimately influencethe signal to noise ratio, either by increasing the signal strength,and/or reducing the noise. Several prior art approaches have beenattempted.

Many common methods used to manufacture high density microarrays usenon-porous, two-dimensional glass substrates containing functional sitesfor binding samples of interest. Glass is preferred because of itsinertness and low inherent auto-fluorescence which contributes lessnoise to the signal being detected, usually measured byfluorescence-based techniques. Examples of such commercially availablesubstrates are UltraGAPS II® slides (Corning Inc., Life Sciences,Oneonta, N.Y.), Nexterion® Slides (Schott North America, Inc.,Louisville, Ky.), and Array-It® slides (Telechem International Inc.,Sunnyvale, Calif.). One drawback to using non-porous glass is that thefunctional site density is quite low resulting in relatively weaksignals, which makes it very difficult to detect the sample of interest,especially when trying to detect low expressing genes or proteins. Thiseffect can be minimized by increasing the volume or concentration of thesample of interest, however, the approach can only be employed if alarge enough sample is available. Often, researchers are highly limitedby the quantity, concentration or volume of a given sample. A commonapproach to increasing functional site density has been through the useof porous substrates to increase the accessible surface area containingthe functional sites. Tanner et al. (U.S. Pat. No. 6,750,023) teach amethod of forming a functional material for attaching an array ofbiological or chemical analytes by applying an inorganic porous layer toan inorganic non-porous understructure.

Alternate approaches using organic polymers as functional materials havebeen attempted. Haddad et al. (WO 01/66244) teach making arraysutilizing textured non-porous functional materials created from orientedpolymer films. Porous organic polymers have also been used in microarraysubstrates and examples of such commercially available materials areVivid Microarray® Slides (Pall Corporation, East Hills, N.Y.) and CAST®slides (Schleicher & Schuell Biosciences, Inc., Keene, N.H.), both usingporous nylon membranes.

Phase inversion is a common technique used to make microporous membranesfrom organic polymers. Use of such membranes as microarray substrates isdescribed in detail in U.S. Patent Applications 2003/0219816 of Solomonet al and 2004/0157320 of Andreoli et al. A variety of microporousmaterials are discussed in the literature, with nylon and nitrocellulosebeing the most common. Nylon affords the benefits that it can be readilyrendered microporous and has a natural affinity for DNA. Similarly,nitrocellulose is known to be effective in binding proteins. In the caseof nylon and nitrocellulose, binding with DNA and/or proteins is relianton the inherent functional groups present in the nylon or nitrocellulosepolymer backbone. Consequently, the functional site density afforded bythese materials is limited. Moreover, the pore size of phase inversionmembranes may not be small enough to prevent lateral spot spreadingwhich leads to crosstalk thereby limiting the array density. Anothercommon problem with using organic polymers such as nylon ornitrocellulose resides with the fact that these materials possessinherently high auto-fluorescence. Since fluorescence-based detection isthe most commonly used technique to quantify the hybridized targetbiomolecules, high auto-fluorescence contributes to increased backgroundnoise thereby adversely affecting the clarity of the fluorescent signal.Use of pigments such as carbon black has been shown to reduce theauto-fluorescence. Alternatively, as taught by Montagu (WO 2004/018623),the background noise can also be reduced by the use of a thin (less thanabout 5μ) functional material.

The need exists for an array substrate that can be easily fabricated,provides high functional site density and exhibits low auto-fluorescenceto maximize signal to noise ratio. The present invention addresses allof these needs along with providing very high level of precision.

SUMMARY OF THE INVENTION

This invention provides a substrate comprising a microporousmicrostructure, an interlayer over at least a portion of themicrostructure and a functional layer attached to the interlayer, thefunctional layer having functional sites with a density of at least 50nanomoles/cm².

In another aspect, this invention provides a method of creating afunctionalized article comprising the steps of (1) providing amicroporous substrate having a microstructure, (2) depositing aninterlayer over the microstructure, (3) and attaching a functional layerto the interlayer such that the article has a functional site density ofat least 50 nanomoles/cm².

In yet another aspect, this invention provides an article comprising asupport layer adjacent to a polytetrafluoroethylene substrate comprisinga porous microstructure, an interlayer over at least a portion of themicrostructure and a functional layer attached to the interlayer, thefunctional layer having functional sites with a density of at least 50nanomoles/cm².

In still another aspect, this invention provides an article comprising asupport layer adjacent to a polytetrafluoroethylene substrate comprisinga porous microstructure, an interlayer over at least a portion of themicrostructure and a functional layer attached to the interlayer, thefunctional layer having functional sites with a density of at least 50nanomoles/cm², and a biomolecule attached to the functional material.

In a further aspect, this invention provides a method of measuringbiomolecules comprising the steps of:

-   -   (a) providing a support layer,    -   (b) functionalizing the support layer,    -   (c) disposing an adhesive on at least part of the support layer,    -   (d) attaching a microporous polytetrafluoroethylene substrate        having a node and fibril microstructure to the support layer via        the adhesive,    -   (e) functionalizing the microporous polytetrafluoroethylene        substrate to form functional sites,    -   (f) binding biomolecules to the functional sites, and    -   (g) detecting the amount of biomolecules bound to the        functionalized layer.

In a further aspect, this invention provides a method of preparing amicroarray substrate comprising

-   -   (a) providing a support layer,    -   (b) optionally functionalizing said support layer,    -   (c) disposing an adhesive on at least a part of said support        layer,    -   (d) attaching a microporous polytetrafluoroethylene substrate        having a node and fibril microstructure to said support layer        via said adhesive, and    -   e) functionalizing said microporous polytetrafluoroethylene        substrate.

In another aspect, this invention provides a microarray substratecomprising an auto-fluorescence level less than 100 RFU at a wavelengthof 635 μm and a functional site density greater than 50 nanomoles/cm².

In another aspect, this invention provides a microarray substratecomprising an auto-fluorescence level less than 1000 RFU at a wavelengthof 532 μm and a functional site density greater than 50 nanomoles/cm².

In another aspect, this invention provides a microarray substratecomprising a signal to noise ratio for the Cy5 dye greater than 130,preferably greater than 150.

In another aspect, this invention provides a microarray substratecomprising a signal to noise ratio for the Cy3 dye is greater than 90,preferably greater than 110.

In another aspect, this invention provides a microarray substratecomprising a 1.5 fold precision level of at least 99%.

In another aspect, this invention provides a microarray substratecomprising a 1.2 fold precision level of at least 76%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a transverse cross-sectional view of one end of an exemplaryembodiment of the present invention.

FIG. 2(A) is a scanning electron micrograph of the microporous surfaceof a microarray of an exemplary embodiment of the present inventionprior to being functionalized.

FIG. 2(B) is a scanning electron micrograph of the microporous surfaceof a microarray of an exemplary embodiment of the present inventionsubsequent to being functionalized.

FIG. 2(C) is a scanning electron micrograph of the microporous surfaceof a microarray of an exemplary embodiment of the present inventionsubsequent to being functionalized.

FIG. 3(A) is a scatter plot of normalized Cy3 and Cy5 signal intensityof microarrays using the substrate of an exemplary embodiment of thepresent invention.

FIG. 3(B) is a scatter plot of normalized Cy3 and Cy5 signal intensityof microarrays using a substrate of the prior art.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed toward improved functional porous andmicroporous substrates with high functional group density and lowauto-fluorescence which when used as a microarray substrate forbio-analytical detection provide heretofore unobtainable highsignal-to-noise ratios with high precision level. These attributesderive from the unique combination of the selection of the porous andmicroporous materials and a method for functionalizing these materials.A microarray can be defined as a tool used to sift through and analyzethe information contained within a genome. This tool contains differentbio-molecular (nucleic acid, protein, cell, etc) probes that arechemically attached to a substrate, which can be a microchip, a glassslide or a microsphere-size bead. In the following discussion, “porousmaterial” refers to a material with pores that extend through the entirecross-section thereby making the material permeable to fluids. Porousmaterials are typically characterized by the mean flow pore size.Alternatively, porous materials can be characterized by bubble pointwhich is a measure of the maximum pore size. Both the mean flow poresize as well the bubble point can be measured by pressure-flow tests.Microporous materials are a subset of porous materials where the meanflow pore size is less than about 1 μm or the bubble point is greaterthan about 10 psi.

Porous materials of this invention are planar in nature and can be inthe form of membranes or sheets. The porous substrates of this inventionare permeable to fluids due to the presence of interconnecting poresthat traverse the entire cross-section. The surface area of thismicrostructure is considerably higher than that of a non-porous materialof equal volume. The present invention utilizes this microstructure andits attendant high surface area to volume ratio in creating the highfunctional density substrate. This internal surface area, betterreferred to as specific surface area, is related to the pore size of theporous material; the surface area increases as the average pore size ofthe material decreases. Typically, the specific surface area of theporous material is at least 0.1 m²/gm and preferably greater than 1m²/gm and most preferably greater than 10 m²/gm as measured by standardgas adsorption techniques.

A “functional site” as used herein is a site located at either anexternal or internal surface of the porous substrate. Functional sitesmay be generated using the surface modification techniques describedherein, and are useful for providing binding sites to which biomoleculesmay be attached. In certain preferred embodiments, the biomolecules thatare attached to the functional sites serve as probe molecules to which atarget biomolecule (typically an analyte in solution) can be bound,either covalently or non-covalently. Non-limiting examples ofbiomolecules contemplated by the invention include nucleic acids,oligonucleotides, and antibodies. “Functional group” as used herein is agroup of atoms that reacts as a single unit and determines theproperties of the functional site. A functional substrate is a poroussubstrate which has functional sites residing on the surface of itsmicrostructure. The term “functionalize” refers to the process in whicha functional group or groups is attached to the microstructure of aporous substrate.

Porous materials with their inherent high specific surface area tovolume ratios offer more area for functionalization than non-poroussubstrates, such as non-porous glass. As mentioned earlier, use of suchfunctional porous substrates for microarray applications have beentaught in the prior art literature such as in US Patent Application2004/0157320 to Andreoli and to U.S. Pat. No. 6,750,023 to Tanner.Although these teachings take advantage of the increased internalsurface area afforded by the porous microstructure, they rely on theinherent functional group density of the porous material for bindingsites. For example, Andreoli teaches the use of porous nylon as thesubstrate with the functionality provided by the amide groups within thechemical structure of the nylon molecule. In comparison, Tanner teachesthe use of porous glass but relies on the presence of surface hydroxylgroup on the glass surface for subsequent functionalization throughsilane treatment.

The present invention takes a novel approach in creating the highfunctional density substrate for microarray application. The inventiveapproach starts with a porous material that does not rely on theinherent chemical nature of the material for creating the functionalgroups. Instead, the microstructure of the porous material issubstantially coated with an intermediate layer containing a reactivefunctionality, such as hydroxyl functionality. The functional substrateis then created by reacting appropriate functional chemistries with thehydroxyl functionality of the intermediate layer. Functionalizing thesubstrate may thus include the step of depositing an interlayer over theporous micro-structure. In this approach, choice of the intermediatelayer and not of the porous material, now controls the density of thefunctional groups. All prior art materials that were subsequentlyfunctionalized in accordance with these teachings of the presentinvention exhibited much higher functional group density.

The high functional density substrate of the present invention isobtained by starting with a porous material, preferably in a planar formsuch as membranes or sheets. The porous materials can be organic orinorganic in nature. Non-limiting examples of such organic porousmaterials could be porous sheets of ultahighmolecular weightpolyethylene (UHMWPE) sold by Porex Corporation, polypropylene (PP) orpolytetrafluoroethylene (PTFE) available from Small Parts, Incorporated(Miami Lakes, Fla.). Membranes made from organic polymers are typicallymicroporous in nature and are available commercially. Examples of suchmaterials are expanded PTFE (ePTFE) membranes available from W. L. Goreand Associates, nylon and polyvinylidenefluoride (PVDF) membranesavailable from Pall Corp under Biodyne™ and Biotrace™ brand namesrespectively, PP membrane available from Osmonics Inc. under PolySep™brandname and PTFE membrane from Porex Corporation under Mupor™ brandname. Inorganic porous materials are typically available as rigidsheets. Such materials typically are obtained by sintering inorganicmaterials such as metals, ceramics and metal oxides. Porous glass is acommon example of such sintered material and is available from companiessuch as R&H Filter company (Georgetown, Del.) or Advanced Glass &Ceramics (Holden, Mass.). Through proper choice of the porous materialand subsequent functionalizing chemicals, the high functional densitysubstrate of the present invention can also be made to have low autofluorescence. Generally, materials devoid of conjugated bonds in theirchemical structure exhibit low fluorescing properties. Examples of suchporous materials are those that are made from materials such as PTFE,UHMWPE, PP, and glass.

Expanded PTFE (ePTFE) is particularly preferred as the porous materialbecause of its low auto-fluorescence as well as for its chemicalinertness and high temperature stability. Methods for making ePTFE aredescribed in U.S. Pat. No. 3,953,566 to Gore. Expanded PTFE is amicroporous form of PTFE consisting of irregular shaped pores. Whereasthe exceptionally high surface area to volume ratio of microporousexpanded PTFE (ePTFE) suggests that it might serve well in thisapplication, the irregular pore shape makes it an unlikely candidate.Surprisingly, however, the irregularity of the ePTFE structure, with itsnon-circular pores, does not compromise the performance; indeed, ePTFEis the most preferred porous material. The pores of expanded PTFE arecreated by an expansion-by-stretching process performed at elevatedtemperatures. Expansion creates a microporous structure in which nodesare interconnected by fine fibrils. Preferred ePTFE materials are madein accordance with the teachings of U.S. Pat. No. 4,187,390 to Gore.

The choice of pore size is a key factor in selecting the porousmaterial. In order to be an effective microarray substrate, the poresmust be small enough to inhibit lateral spreading of the solution duringthe spotting process. It is believed that if the pores are too large,the spotting liquid will spread in all directions and the spots will runinto each other thereby leading to crosstalk and contamination. The spotto spot distance can be increased to avoid this problem, however, thiscompromises the number of spots that can be placed within a given area.On the other hand, the pores must be large enough to enable thebio-molecules to enter the pores and to allow reagents to enter and exitthe pores freely during washing processes. Bubble point measurement is astandard technique to characterize the maximum pore size of porousmaterials.

Whereas most porous substrates can be used to create the high functionaldensity substrate of the present invention, for microarray applications,the bubble point should be at least 0.007 MPa (1 psi), preferably atleast 0.070 MPa (10 psi). Preferred ePTFE materials possess bubble pointvalues of at least 0.070 MPa (10 psi). Most preferably, the ePTFEmaterial has bubble point values of at least 0.207 MPa (30 psi).

The microstructure of microporous ePTFE material consists of nodesinterconnected by fibrils and can be characterized by its average fibrillength. The fibril length can be measured by taking a scanning electronmicrograph of the surface of the ePTFE membrane at reasonably highmagnification (such as at 20,000×) and then measuring the length of thefibrils between the nodes. Thirty such measurements are taken of fibrilsand the average fibril length is reported as the average of thesemeasurements. Larger average fibril lengths are typically associatedwith lower bubble point and higher mean flow pore size. For thepreferred ePTFE materials, it is believed that the average fibril lengthshould be between 0.5 to 5 μm, preferably between 0.5 to 3 μm and mostpreferably between 0.5 to 2 μm.

The high functional site density substrate of this invention can beformed by using a porous material ranging in thickness from 5 μm andabove. Increased thickness provides higher internal surface forattachment of functional groups thereby leading to increased functionaldensity. For microarray applications, however, there is a limit to thethickness of the porous material. An excessively thick material is notdesirable since such a material may absorb excessive amounts of probesolution during contact printing thereby causing the printing pins torapidly become dry, thus affecting the spot clarity. In addition,excessively thick materials are difficult to process particularly duringthe washing step after hybridization. Inadequate washing of thehybridization liquids from the material can lead to residual reagentscausing increased auto-fluorescence of the substrate. For a microarraysubstrate, the preferred thickness of the porous material is about 250μm or less, most preferably about 125 μm or less.

The present invention relies on the internal surface of the porousmaterial to attach the functional group. Internal surface area is afunction of the thickness and the specific surface area of the porousmaterial. Expectedly, specific surface area is an importantconsideration for selection of the porous material. However, specificsurface area is related to the pore size. Typically, the smaller thepore size, the greater the specific surface area. Consistent with poresize requirements, porous material with any specific surface area can beconverted into a high functional site density of this invention.However, for a viable microarray substrate, the porous material shouldpossess a specific surface area of at least 1 m²/gm. The preferred ePTFEmaterial has at least a 1 m²/gm of specific surface area and mostpreferably at least 10 m²/gm of specific surface area.

The porous material that can be used for the present invention ispreferred to be free of any additives, particularly additives that cancontribute to increased auto-fluorescence. However, if needed, theporous material can contain additives such as pigments, fillers,colorants, UV absorbers and the like.

The porous materials are converted into high functional density poroussubstrates of this invention by first depositing an intermediate layerof hydroxyl containing functional coating on the entire microstructureof the porous material and subsequently using organosilane chemistry toreact with the hydroxyl group of the intermediate layer. Details of thisconversion process are described below primarily for the preferred ePTFEmaterial. However, the conversion method can similarly be applied to alarge variety of porous materials described in the preceding sections.

Because of the inherent hydrophobicity of ePTFE, polar solutions such asmicroarray printing and hybridization buffers do not wet the substratematerial. Also, due to the chemical structure of ePTFE, bio-moleculessuch as nucleic acids or proteins do not efficiently bind to thematerial. Consequently, for effective binding of biomolecules, the ePTFEsurface must be modified and functional groups need to be subsequentlyattached. Surface modification of ePTFE by coating its microstructureusing organic polymers in order to render its surface hydrophilic hasbeen described in U.S. Pat. Nos. 5,130,024 and 5,897,955 to Fujimoto andto Drumheller, respectively. Similar hydrophilic treatment of ePTFEusing inorganic sol-gel formulations has been described in Japanesepatent publication number 08-250101 and in US Patent application2004/0081886A1 to Zuckerbrod.

For the present invention, it is preferred that the surface modificationbe performed using low auto-fluorescing hydrophilic coatings thatprovide hydroxyl groups capable of subsequent reaction with silanes.Non-limiting examples of organic polymers that are suitable for suchhydrophilic coatings are polyvinyl alcohol, polyethyleneglycol,polypropylene glycol, polyglycidol, poly(vinyl alcohol-co-ethylene),poly(ethyleneglycol-co-propyleneglycol),poly(ethyleneglycol-co-propyleneglycol), poly(vinyl acetate-co-vinylalcohol, either alone or in combination. Optionally, these polymericcoatings can be covalently cross-linked to themselves in situ by usingsuitable cross-linking agents such as aldehydes, epoxides, anhydridesetc. Polyvinyl alcohol (PVOH) is the preferred organic polymer for thehydrophilic treatment of ePTFE. The optional cross-linking can beachieved by the use of aldehydes such as glutaraldehyde.

Sol-gel solution, as described below, is a more preferred solutioninasmuch as it renders the ePTFE surface more amenable for subsequentfunctionalization by providing a larger number of hydroxyl groups.Sol-gel is a technique for preparing specialty metal oxide glasses andceramics by hydrolyzing chemical intermediates or mixtures of chemicalintermediates that pass sequentially through a solution state and a gelstate before being dehydrated to a glass or ceramic. The details of thesol-gel treatment used to make ePTFE hydrophilic are described in theJapanese patent publication number 08-250101.

The preferred sol-gel coating solution is derived fromtetraethylorthosilicate (TEOS), tetramethylorthosilicate, or a sol-gelcoating derived from sodium silicate solution or a colloidal silicasuspension. Sol-gel coating solution derived from TEOS is the mostpreferred. Hydrophilic coatings, described above, can also be used tosurface modify other microporous materials including but not limited tothose made from nylon, ultrahigh molecular weight polyethylene (UHMWPE),polypropylene, porous PTFE, PVDF, porous glass, and the like. Thehydrophilic treatment of the ePTFE and other microporous materials canbe achieved by a number of ways. Usually, this treatment is achieved byapplying a solution of the organic polymer or the inorganic sol-gel tomembranes by commonly known methods such as dip coating, spraying, spincoating, brushing, roller coating, or Meyer bar coating. Care must betaken to add only enough of the coating to render the surfacehydrophilic while maintaining the porosity of the material. Addingexcessive amounts of the hydrophilic coating also will increase theauto-fluorescence of the substrate.

The hydrophilic treatment step, described above, renders the ePTFE andother microporous materials hydrophilic by depositing a hydroxylcontaining coating over the entire microstructure. In a subsequent step,the hydroxyl groups are reacted with low auto-fluorescing organosilanesto obtain the desired functional groups depending on the specificbiomolecules to be attached. For example, if complementary DNA (cDNA)molecules are to be attached to the substrate, amine functionality ismost suitable and such functionality can be introduced by reacting thehydrophilized ePTFE material with suitable straight or branchedaminosilane, aminoalkoxysilane, aminoalkylsilane, aminoarylsilane.Examples of silanes that can be used are γ-aminopropyltrimethoxysilane,γ-aminopropyltriethoxysilane,N-(beta-aminoethyl)-γ-aminopropyltrimethoxysilane,N-(beta-aminoethyl)-γ-aminopropyltriethoxysilane. Such aminefunctionality can also be introduced through organosilane coupleddendrimers available from companies such as Dendritech, Inc. (Midland,Mich.). Using this approach, through selection of appropriateorganosilanes, different reactive functional groups can be attached tothe ePTFE substrates. For the same functional group, the placement ofthe functional group from the attachment site on the surface can also becontrolled by the size of the linker molecule used in the organosilaneselected. Non-limiting examples of reactive functional groups that canbe attached are amines, epoxides, adehydes, carboxyls, anhydrides,hydroxyl, acrylates, methacrylates, esters, thiols, azides, sulfonatesand phosphonates to name a few. If desired, more than one functionalgroup can be deposited on the substrate by reacting a mixture of silaneswith different organofunctional groups with the hydroxyl groups from theintermediate layer. The functional groups can be further reacted withother chemical reagents to create the desired functionality for thetargeted end use. For example, epoxide groups can be further reactedwith diols to recreate hydroxyl functionality or amine functional groupscan be further crosslinked through use of maleimide—NHS basedcross-linkers to create a functionality that can react with biomoleculespossessing amine functionality such as antibodies.

The silane treatment can be achieved by treating the hydrophilic ePTFEmaterial with a dilute solution of the silane in an organic solvent atlow pH. Details of such silane treatment and the variety of silanes withdifferent organofunctional groups and different linker size can beobtained from the brochure “Silane Coupling Agents: Connecting AcrossBoundaries” available from Gelest, Inc. of Morrisville, Pa. Similarinformation and chemicals are also available from other companies suchas United Chemical Technologies (Bristol, Pa.), Dow-Corning (Midland,Mich.), and GE Advanced Materials (Wilton, Conn.). For the presentinvention, the silane treatment can be achieved through conventionalliquid coating processes such as dip coating, spraying, spin coating,brushing, roller coating, or Meyer bar coating. Alternatively, thesilane can be deposited on the membrane microstructure through vaporphase coating. Care must be taken to add only enough of the silane tofunctionalize the ePTFE while maintaining the porosity of the material.

The process of the present invention dramatically improves thefunctionality of a variety of membranes as evidenced by significantlyimproved amine density values which can be measured using assaysfamiliar to those skilled in the art. Most remarkably, treated ePTFE andother microporous membranes of the present invention exhibit well overan order of magnitude improvement in amine group density compared toprior art materials. Improvements to functionalized membranes of thismagnitude were surprising and unexpected.

The functional group density can be measured in terms of moles offunctional groups per unit superficial area of the substrate or per unitvolume of the substrate. Using the inventive method, porous materialswith amino group density ranging between 0 and 5 nanomoles/cm² wereconverted to substrates with functional group densities in the range of50 to 1300 nanomoles/cm² depending on the specific chemical nature ofthe porous material. Given the thicknesses of these substrates, thefunctional density per unit volume translates to 2500 to 150,000nanomoles/cm³. For example, ePTFE and microporous PP materials whichwere devoid of any amino functionality were converted into aminofunctional substrates with amino functional density of 416 and 487nanomoles/cm² using the inventive method. Since the inventive methodgenerates functional sites by reacting different organosilanes with thehydroxyl groups deposited during the intermediate layer coating, theabove functional group density values are not limited only to aminefunctional groups. Rather, the functional site density is expected todepend primarily on the density of hydroxyl groups resulting from theintermediate layer coating. It is therefore anticipated that throughsuitable choice of organofunctional silane compounds, functional sitedensity greater than 50 nanomoles/cm² or greater than 2500 nanomoles/cm³can be achieved irrespective of the specific nature of the functionalgroups chosen.

Apart from use in microarray applications, the highly functionalsubstrates of this invention can be used for effective binding andcapture of a large variety of biomolecules in other applications such asin diagnostic devices, active filtration applications, blottingapplications, and the like.

This remarkable increase in functional density of the substrate can beachieved while keeping auto-fluorescence quite low. This combination ofhigh functional density and low auto-fluorescence is highly desirable ina microarray substrate since it maximizes the fluorescence signal fromthe hybridized target biomolecules over the background noise. Theauto-fluorescence of the substrate can be determined by scanning thesubstrate prior to printing biomolecules using microarray scannersavailable commercially from several vendors such as Axon Instruments(Union City, Calif.) and Perkin-Elmer (Wellesley, Mass.). Scanning canbe done at multiple wavelengths of interest, depending on the type ofscanner used. Average fluorescence values can be calculated at thewavelengths of interest from the scanned substrate data. It should benoted that scanning can be performed at different instrument settingssuch as laser power, focus depth and PMT gain. Signal intensities arefunction of these settings. Therefore auto-fluorescence values should beaccompanied by the scanner settings for a given instrument.

A GenePix 4000A scanner (Axon) was used to measure the auto-fluorescenceof the substrates for this invention. The laser power and the focusdepth of this scanner were fixed at 100% and 0 μm respectively, and allmeasurements were done at a PMT setting of 350. Using the method of thisinvention, for low fluorescence porous materials such as PTFE, UHMWPE,PP, glass, etc.; high functional density substrates can be made withauto-fluorescence level less than 1000 relative fluorescence units (RFU)and less than 100 RFU for the 532 nm (green) and 635 nm (red)wavelengths, respectively. In most cases the auto-fluorescence level ofsuch high functional site density substrates can be much lower,typically less than 100 RFU and 30 RFU at 532 nm and 635 nm wavelengths,respectively.

The high functional density and low auto-fluorescence porous substrateis most suitable for microarray applications as it is expected toprovide increased signal intensity over background noise. There arevarious ways such substrates can be used in microarray application. Thesubstrate can be used as is or alternatively the substrate can beconverted into a cassette or into the shape of a microscope slidethrough combining it with other plastic, ceramic or metallic partsthrough insert molding or other assembly techniques such ultrasonicbonding, RF welding, heat welding, or the like. By choosing theappropriate functionality, it is possible to attach large variety ofbiomolecules to the high functional site density substrate of thisinvention. Non-limiting examples of biomolecules that can be attachedare nucleic acids, proteins, peptides, oligonucleotides, antibodies,cells, enzymes, and pathogens, to name a few.

For many applications, in order to ease both handling and printing, itis desirable to support the high density functional site substrate ofthis invention with a support layer. This support layer can be bothflexible as well as rigid. The flexible support layer can be plasticfilms and metal foils. However, for traditional microarray applicationthe support layer is typically rigid. Such rigid support layer can bemade from a stiff material as long as the material maintains dimensionstability at hybridization temperatures and does not get affected by thereagents used during printing, hybridization, washing and drying stepsinvolved in typical microarray experiment. Non-limiting examples ofmaterials that are suitable as the support layer are glass, metals,ceramics, and plastics. A glass microscope slide is most commonly usedas the support layer.

In an embodiment of the present invention, the functional substrate maybe adhesively bonded, at least partially, to the rigid support to createa composite microarray substrate. The resulting adhesive bond should bestrong enough to survive processing steps of printing, hybridization,washing and drying steps involved with a typical microarray experiment.The adhesive therefore needs to possess the appropriate thermal andchemical resistance. It is also desired that the adhesive exhibits aslow an auto-fluorescence level as possible. Typically, adhesivescontaining no conjugated bonds in their chemical structure are likely todemonstrate low auto-fluorescence. The adhesive chosen must bond well tothe rigid support as well as to the functional porous layer. If needed,the support surface can be treated to enhance the bond with theadhesive. Treating the support surface with organosilane is an exampleof a surface treatment that can be used to enhance adhesion.Alternatively, adhesion-promoting additives such as silane couplingagents can be added to the adhesive to promote better bond between theadhesive and the rigid support. For acceptable bond to the functionalporous substrate, the adhesive needs to penetrate into the porousstructure. If the adhesive viscosity or surface tension is not lowenough to allow this penetration, the adhesive can be solvated with lowviscosity and/or low surface tension solvents to promote thispenetration. However, caution must be exercised to ensure that theadhesive does not penetrate excessively into the cross-section of thefunctional substrate since this would reduce the availability of thefunctional groups as well as increase the auto-fluorescence level of thecomposite microarray substrate

Various kinds of adhesive can be used. The adhesive can be thermosettingin nature. Examples of such adhesives include but are not limited toepoxies, acrylics, silicones. These types of adhesives can be appliedeither to the support layer or to the functional layer, contacted to theother surface to be bonded, and then cured through application of energyin the form heat, UV radiation or the like. TRA-BOND FDA2 available fromTra-Con, Incorporated (Bedford, Mass.) is an example of a two-partthermosetting epoxy that can be used. If the adhesive is in liquid form,it can be applied by a variety of commonly used methods such asspraying, brushing, roller coating, etc. If the adhesive is in the formof a partially cured film, it can be laminated through application ofpressure and/or heat. The adhesive can also be pressure sensitive innature and belong to different chemical families. Acrylics (e.g., 3M9461P Adhesive transfer tape from 3M Corporation, St. Paul, Minn.) andsilicones (e.g. Dow-Corning® MD7-4602 from Dow-Corning Corporation,Midland, Mich.) are commonly used pressure sensitive adhesives (PSAs).In this case, the adhesive is applied either to the support or thefunctional substrate and bonded to the other material throughapplication of pressure and/or heat. If the adhesive is in liquid form,it is applied as indicated above, dried if necessary to remove anyvolatiles and then bonded to its counterpart. Finally, the adhesive canbe thermoplastic. Examples of such adhesives with low fluorescingproperties are fluoroplastics (Dyneon™ THV Fluorothermoplastic fromDyneon LLC, Oakdale, Minn.); eFEP™ from Daikin America, Inc.,Orangeburg, N.Y.; Teflon® FEP (Dupont Fluoroproducts, Wilmington, Del.),Topas® cyclic olefin copolymers from Ticona, Chatham, N.J., to name afew. The film form of these materials can be used to bond the functionallayer to the support layer through application of heat and pressure in alamination step. If available in resin form, the thermoplastic materialcan be dissolved in a suitable solvent and applied as a thin layer oneither the functional membrane or the support layer, dried to remove thevolatiles and bonded to its counterpart. The porous material layer canbe adhesively bonded to the support prior to functionalization and thenthe composite can be functionalized through the steps of hydrophilictreatment and silane treatment as discussed previously.

FIG. 1 shows an article 10 according to an exemplary embodiment of thepresent invention. A support layer 12 has an adhesive 14 disposedthereon. A microporous fluoropolymer, ePTFE, substrate layer 16 isattached to support layer 12 by adhesive 14.

Support layer 12 is any rigid surface capable of bonding to microporousfluoropolymer layer 16, with or without the use of an adhesive. Glass ispreferred for support layer 12. The surface of support layer 12 isoptionally treated before adhesive 14 (if used) and microporousfluoropolymer layer 16 are applied.

A scanning electron micrograph of the surface 20 of the ePTFE materialis shown in FIG. 2(A). This figure indicates the presence of nodes 22interconnected by fibrils 24. The photomicrograph also depicts theirregular pores of this material.

A scanning electron micrograph of the surface of the porous substrate ofthis invention using ePTFE as the starting material is shown in FIGS.2(B) and 2(C) at two different magnifications. The microstructure of theporous ePTFE materials was first treated with silica sol-gel to createan intermediate layer which was then reacted with an aminosilane. Thecoated nodes 26 and coated fibrils 28 of the microstructure are shown inFIG. 2(C).

Using the inventive method, composite microarray substrates employingthe functional ePTFE layer were made that exhibit the desired featuresof unusually high functional group density and low auto-fluorescence. Inparticular, depending on the characteristics of the ePTFE, compositesubstrates can be made with functional group density of at least 50nanomoles/cm², preferably of at least 100 nanomoles/cm² and mostpreferably of at least 250 nanomoles/cm². This is at least an order ofmagnitude higher than functional group densities obtained for prior artsubstrates. For example, depending on the specific characteristics ofthe ePTFE material used, the amine densities measured for the compositemicroarray substrates range from about 100 to 400 nanomoles/cm². Incomparison, the amine densities measured for aminosilane treatednon-porous glass slide (Corning Ultragaps™) and porous nylon membranebased Vivid™ microarray slide were about 4.8 and 6.5 nanomoles/cm²respectively.

While providing high functional site density, the inventive compositemicroarray substrate maintains its auto-fluorescence at a low level. Theauto-fluorescence of the composite substrate can be determined byscanning the substrate prior to printing biomolecules using GenePix4000A microarray scanner at a PMT setting of 350. Using the method ofthis invention, a composite microarray substrate comprising a functionalePTFE layer can be made with auto-fluorescence level less than 1000relative fluorescence units (RFU) and less than 100 RFU for the 532 nm(green) and 635 nm wavelengths, respectively. In most cases theauto-fluorescence level of the composite microarray substrates can bemuch lower, typically less than 200 RFU and 30 RFU at 532 nm and 635 nmwavelengths, respectively.

The composite microarray substrate of this invention provides aversatile surface for immobilization of biomolecules. Other than its usein typical microarray analysis, the inventive substrate can also be usedas a substrate with a variety of biomolecules attached to it in anyarbitrary pattern. Examples of biomolecules that can be attached arenucleic acids, proteins, peptides, oligonucleotides, antibodies, cells,pathogens, to name a few.

The performance of the composite microarray substrate was determined byconducting an evaluation in which a DNA microarray was created using thecomposite substrate. The microarray was then hybridized with cDNAlabeled with two fluorescent dyes, namely Cy3 and Cy5, that emitfluorescent signals at two different wavelengths. The fluorescentsignals, at two different wavelengths, from each spot (and its vicinity)within the hybridized slides were then detected using a microarrayscanner using a laser light source and a photo multiplier tube (PMT) asthe detector. The scanner detects the fluorescent light intensities fromthe hybridized microarray substrate and the data is stored in the formof a scanned image of the substrate representing intensities on a colorscale. The raw signal intensity data, thus obtained, were statisticallyanalyzed using standard microarray data analysis software (such asGenePix® Pro from Axon Instruments, Genetraffic® from Lobion informaticsor Scanarray® Express from Perkin-Elmer) to determine some keyperformance metrics such as signal to noise ratio and precision level.These performance criteria were determined for the composite microarraysubstrate of this invention as well as for substrates representing theprior art. Details of microarray data analysis are readily available inbooks such as “DNA Microarrays”, edited by Ulrike A Nuber, Taylor &Francis, N.Y., 2005 or “Microarray analysis” by Mark Schena, John Wiley& Sons, Hoboken, N.Y., 2003.

Signal to noise ratio (SNR) is a key performance measure for amicroarray substrate. The quality of the signal from a spot within amicroarray depends on its intensity relative to its immediatesurroundings, also known as local background noise. As the signalintensity from a spot approaches the intensity of the local backgroundnoise, the error in each measurement becomes potentially higher. At agiven wavelength, the SNR for a spot can be easily computed by firstdetermining the net signal intensity, which is the difference betweenthe median signal intensity (S) for all pixels representing a spot andits median local background (B) for all pixels representing theimmediate area just outside the spot. The background noise (NB) isestimated by calculating the standard deviation of the local background.SNR for the spot is then defined as:SNR=(S−B)/NBin which S, B, and NB are expressed in relative fluorescence units(RFU).

Typically, the SNR is determined for individual spots in an array. Theaverage SNR (ASNR) is the average of all the SNR for individual spots inan array. Using microarray data analysis software, SNR calculations canbe performed automatically for the large number of spots within atypical microarray.

High ASNR is always desired since it provides higher confidence in theaccuracy of the data obtained from a microarray experiment. Thecomposite microarray substrate of this invention provides remarkablyhigh ASNR as compared to substrates of the prior art. In general, onaverage, the inventive composite substrate exhibits ASNR which is atleast twice that obtained from aminosilane treated glass slides at bothof the wavelengths. For example, the performance of a functionalizedePTFE membrane adhered to a glass slide far exceeds that of all priorart materials. It exhibits average signal to noise ratios for Cy5 andCy3 of at least about 191 and 94, respectively. The most commonly usedprior art slide exhibits signal to noise ratios for Cy5 and Cy3 of about110 and 62, respectively

It was surprising to notice that the composite microarray substrate ofthe present invention not only provides high ASNR, but was also veryeffective in stabilizing the fluorescent signal obtained. It is wellknown in the art that the signal from Cy5 dye is extremely unstableparticularly under the influence of ozone. In fact, due to seasonalvariation in ozone level in the ambient, it is not unusual to see thestability of the Cy5 signal deteriorate when the ambient ozone levelsincrease. It has now been found that the composite microarray substrateof this invention employing functional ePTFE layer is remarkably moreeffective in stabilizing the Cy5 signal as compared to that seen onsubstrates of the prior art. For example, in summer months when ambientozone levels were high, the Cy5 SNR for the inventive substrate wasabout 7.7 times that of the Cy5 SNR on aminosilane treated glass slideand this ratio. Within 24 hours, this ratio increased to 38.9 as the Cy5signal on the glass slide reduced drastically whereas the Cy5 signal wasrelatively more stable on the composite substrate of this invention.

In addition to high ASNR, precision is another performance measure thatis highly desirable. In a microarray experiment, when the same target islabeled with two different fluorescent dyes (Cy3 & Cy5); it is expectedthat signals from both the wavelengths should provide the sameinformation. In other words, if the Cy3 signal intensity is plottedagainst the Cy5 signal intensity on a graph with identical x and y axes,ideal data should lie on the 1:1 (or 45 degree) line. In reality, thedata generally deviates from this line and the further this deviation isfrom the 1:1 line, the less reliable the data becomes. A measure of theprecision of the data can be obtained by devising a measure of how closethe data are to the 1:1 line. If there are M number of data points andout of that set if N data points lie outside the Z-fold up and Z-folddown boundaries, the Z-fold precision level can be defined asP _(Z) =Z-fold % Precision level=100×(1−(N/M))in which Z-fold up and Z-fold down boundaries represent relationshipswhere the Cy3 signal intensity is Z times or 1/Z times that of the Cy5signal intensity respectively. For example, 2-fold up implies that theCy3 signal intensity is twice that of the Cy5 signal intensity and2-fold down implies that the Cy3 signal intensity is half that of theCy5 signal intensity. Higher P_(Z) values at lower Z levels indicatemore precise and reliable data. The composite microarray substrate ofthe present invention exhibits remarkably high precision level.Typically, P_(1.5) and P_(1.2) were at least 99% and at least 90%,respectively, for the substrates of this invention. In comparison,respective values for aminosilane treated glass slide were 96 and 73%,respectively. Clearly, the composite substrate described here yieldsextremely precise and reliable data when used in a microarrayexperiment.

EXAMPLES

Test Methods

Thickness Measurement

Membrane thickness was measured by placing the membrane between the twoplates of a Kafer FZ1000/30 thickness snap gauge (Käfer MessuhrenfabrikGmbH, Villingen-Schwenningen, Germany). The average of the threemeasurements was used.

Bubble Point Measurement

The bubble point and mean flow pore size were measured according to thegeneral teachings of ASTM F31 6-03 using a Capillary Flow Porometer(Model CFP 1500 AEXL, Porous Materials Inc., Ithaca, N.Y.). The samplemembrane was placed into the sample chamber and wet with SilWickSilicone Fluid (Porous Materials Inc., Ithaca, N.Y.) having a surfacetension of 19.1 dynes/cm. The bottom clamp of the sample chamber had a2.54 cm diameter, 3.175 mm thick porous metal disc insert (40 micronporous metal disk, Mott Metallurgical, Farmington, Conn.,) and the topclamp of the sample chamber had a 3.175 mm diameter hole. Using theCapwin software (version 6.62.1) the following parameters were set asspecified in the table immediately below. The values presented forbubble point and mean flow pore size were the average of twomeasurements.

Parameter Set Point Parameter Set Point maxflow (cc/m) 200000 mineqtime(sec) 30 bublflow (cc/m) 100 presslew (cts) 10 F/PT (old bubltime) 40flowslew (cts) 50 minbppres (PSI) 0 eqiter 3 zerotime (sec) 1 aveiter 20v2incr (cts) 10 maxpdif (PSI) 0.1 preginc (cts) 1 maxfdif (cc/m) 50pulse delay (sec) 2 sartp (PSI) 1 maxpre (PSI) 500 sartf (cc/m) 500pulse width (sec) 0.2Functional Group Density Measurement

A ninhydrin based assay was used to determine the density of thefunctional amino groups. The assay was based on the teachings of Sarinet. al. (Sarin, V. K., Kent, S. B. H., Tam, J. P. & Merrifield, R. B.(1981) Anal. Biochem. 117, 147-157). In this assay, ninhydrin wasreacted with the substrates of this invention. The reaction productwithin the resulting liquid was sprectroscopically determined to arriveat the concentration of amine functionality. The assay used about 1 cm²size specimens obtained from the sample substrates and the followingprocedure was employed:

Reagent A—In a beaker, 40 g of phenol and 10 ml of absolute ethanol weremixed and warmed until a clear liquid was obtained. In a separatebeaker, 0.042 g of potassium cyanide (KCN) was dissolved in 65 ml ofwater. Approximately 2 ml of this KCN solution was then diluted with 100ml of absolute pyridine in a separate bottle. In a separate containerlabeled “Reagent A,” 6 ml of the phenol/ethanol solution was mixed with12.5 ml of the KCN/pyridine solution.

Reagent B—2.5 g of ninhydrin was dissolved in 10 ml of absolute ethanol.

Sample Analysis

In a test tube, 800 μl of Reagent A and 200 μl of Reagent B were added.The test tube was placed in a heating block set at 100° C. and the blockwas placed over a shaker. The shaker was run at 110 rpm for 10 min. Thetest tube was then removed and placed in a water bath. Ethanol was addedto the tube until the total volume was 2 ml and the solution waswell-mixed. 200 μl aliquots of this mix were pipetted into a glass 96well plate and the absorbance at 570 nm was measured using aspectrophotometer.

Data Analysis—

The amine density for each sample was calculated from the followingrelationship using the absorbance value after the blank absorbance wassubtracted out.Amine Density (nanomoles/cm²)=[Absorbance_(sample)*Volume (L)*10⁹(nmol/mol)]/[Ext.Co.₅₇₀ (M⁻¹ cm⁻¹)*Pathlength (cm)*Area_(sample) (cm²)],in which Volume=2 ml=0.002 L, Ext. Co.=Extinction Coefficient=15,000 M⁻¹cm^(−.), and the pathlength used was 0.4146 cm.

For each sample, three measurements were made and the amine densityvalue was reported as the average of the three replicates.

In the case of unsupported functional substrates, the thickness of thesubstrate was directly measured. In this case, the functional groupdensity was expressed as:Functional group density (nanomoles/cm³)=functional group density(nanomoles/cm²)/substrate thickness (cm)Auto-Fluorescence Measurement

The auto-fluorescence of the unsupported substrates and the microarrayslides prepared with these functionalized substrates was measured usingan Axon Genepix 4000A (Axon Instruments Inc., Union City, Calif.)scanner with a PMT setting of 350 and a resolution of 10 μm. Autofluorescence was measured at wavelengths of 635 nm and 532 nm. Slidessamples (including rigid Vycor substrates) were placed in the slideholder with the substrate facing down and scanned for auto fluorescence.In the case of unsupported substrates such as membranes, the sampleswere draped around a plain glass microscope slide and placed in theslide holder with the substrate facing down and scanned for autofluorescence. The scanned image was analyzed using GenePix Pro 5.0software. Auto fluorescence values were recorded at 4800 discretelocations within the slide over a rectangular area. The top left cornerof this area was 2.27 mm from the left edge and 12.21 mm from the topedge of the 25.4 mm×76.2 mm sample. The bottom right hand corner of thisarea was 18.04 mm from the left edge and 59.82 mm from the bottom edgeof the sample. The average auto fluorescence values at the twowavelengths were reported for each sample slide and each substrate.

Signal to Noise Measurement

Signal to noise measurements of the microarray slides for the presentinvention were conducted by the Microarray Centre at University HealthNetwork (UHN), Toronto, Canada. A 1718 clone set from the human genomewas printed on the slide using a printing solution of the DNA in 3×SSCat a concentration of 0.2 μg/ml. The printed array was organized in 32blocks arranged in 8 rows and 4 columns with a grid-to-grid distance of4500μ. Within each grid, there were 120 features arranged in 10 rows and12 columns. The feature size was 100 μm and the feature-to-featuredistance was 200μ. Humidity level during printing was controlled between55-60%. Following printing, the printed probes on the slide were driedat 95° C. for 1 minute and then cross-linked at 2500 micro Joules ofpower using a UV Stratalinker™ 1800 (Stratagene).

The following labeling protocol was used to generate labeled cDNA from10 μg total RNA.

Reverse Transcription

-   -   In a 0.5 μl tube, combine 8.0 μl of 5× First Strand buffer        (Superscript II, Invitrogen), 1.5 μl of AncT primer (5′-T₂₀VN,        100 pmol/μl), 3.0 μl of dNTP-dTTP (6.67 mM each of dATP, dCTP,        dGTP), 3.0 μl of 2 mM dTTP, 3.0 μl of 2 mM AA-dUTP (Sigma,        catalog no. A-0410), 4.0 μl of 0.1 M DTT, 1.0 μl of control RNA        (artificial Arabadopsis transcripts (2-10 ng/μl), optional),        0.1-10 μg of total RNA (0.1-0.5 μg mRNA or 5-10 μg total RNA),        and 40 μl of nuclease-free water.    -   Incubate the labeling reaction at 65° C. for 5 minutes, then at        42° C. for 2 minutes (to partially cool solution). It is not        necessary for the incubation to occur in the dark.    -   Add 2 μl reverse transcriptase (Superscript II, Invitrogen) and        incubate at 42° C. for 2 hours.    -   Add 8 μl of 1M sodium hydroxide and heat to 65° C. for 15        minutes to hydrolyze RNA.    -   Add 8 μl of 1M hydrochloric acid and 4 μl of 1M tris-HCL, pH 7.5        to neutralize the solution.        Amino Allyl-cDNA Purification

Purification was performed using CyScribe™ GFX™ Purification kit (GEAmersham, catalog no. 27-9606-02). Each sample was purified in one GFXcolumn, using the following protocol.

-   -   Add 500 μl of capture buffer to each column.    -   Transfer cDNA product (approx 62 μl) to the column, pipette up        and down several times to mix, spin at 13800×g for 30 seconds        and discard flow-through.    -   Add 600 μl of 80% ethanol and spin at 1300 rpm for 30 seconds        and discard flow-through; repeat this step for a total of 3        washes.    -   Spin the column for an additional 30 seconds to ensure all        ethanol is removed.    -   Transfer the GFX column to a fresh tube and add 60 μl of 0.017 M        sodium bicarbonate, pH 9.    -   Incubate the GFX column at room temperature for 1 minute.    -   Spin at 13800×g for 1 minute to elute purified labelled cDNA.    -   Use Speed Vac to completely dry sample. Resuspend in 7 μl        nuclease-free water.\

Preparing Monofunctional Reactive Cyanine Dye & Labeling

-   -   Alexa 647/Alexa 555 fluors (Invitrogen) and Cy5/Cy3 (Amersham)        were used in this study and both will be referred to as Cy5 &        Cy3 respectively. The Alexa fluors are sold individually        packaged. Add 3 μl of DMSO per tube to resuspend the dye. Add        entire contents of the tube to each labeling reaction. The Cy        dyes come in packages of 5 μl. Add 45 μl of DMSO to each tube.        Again, 3 μl of the resuspended dye was added to each labeling        reaction.    -   Add 3 μl dye to 7 μl aminoallyl-labelled cDNA, mix by pipetting        up and down, and incubate in the dark at room temperature for 1        hour.    -   Add 4.5 μl of 4M hydroxylamine to quench non-conjugated dye.        Incubate in the dark at room temperature for 15 minutes.        Purification of Fluorescent Labeled Probe    -   Add 35 μl water to each reaction to bring each reaction volume        to about 50 μl.    -   Combine the Cy5 and Cy3-labelled samples that will be        co-hybridized.    -   Add 500 μL of capture buffer to each column.    -   Transfer labelled-cDNA product (approx. 100 μL) to the column,        pipette up and down several times to mix, spin at 13,800×g for        30 seconds and discard flow-through.    -   Add 600 μL 80% ethanol and spin at 13,800×g for 30 seconds and        discard flow-through; repeat this step for a total of 3 washes.    -   Spin the column for an additional 30 seconds to ensure all        ethanol is removed.    -   Transfer the GFX column to a fresh tube and add 60 μL elution        buffer (provided with kit)    -   Incubate the GFX column at room temperature for 1 minute.    -   Spin at 13,800×g for 1 minute to elute purified fluor-labelled        cDNA.    -   SpeedVac sample to dryness (on high heat; be careful not to        over-dry) and resuspend in 5 μL nuclease-free water        Hybridization    -   A prehybridization step is not required.    -   Make enough solution for all your hybridizations—make 100 μl per        slide and an additional 100 μl for pipetting error.    -   To each 100 μL of DIG Easy Hyb solution (Roche), add 5 μL of        yeast tRNA (Invitrogen; 10 mg/ml) and 5 μL of calf thymus DNA        (Sigma; 10 mg/ml). Incubate the mixture at 65° C. for 2 minutes        and cool to room temperature.    -   Add 100 μl of the prepared hybridization solution to each pooled        pair of Cy5 and Cy3-labelled cDNA (about 5 μL).    -   Mix the hybridization solution with the labelled-cDNA, incubate        at 65° C. for 2 minutes, and cool to room temperature    -   Place cover slip (24×60 mm, non-lifter slip) onto a reliable        surface (the corner of a tip box works well) and pipette the        hybridization mixture onto the cover slip. Lay the slide        “array-side” down on top of the cover slip (do not actually put        the slide down on the cover slip simply hold it on top of the        cover slip until the slide is wetted enough to pick up the cover        slip). Quickly flip the slide, with cover slip stuck to it, over        so the cover slip is on top of the slide.    -   Carefully place the slide(s) into hybridization chamber(s). The        hybridization chambers that we use are plastic microscope slide        boxes containing a small amount of DIG Easy Hyb solution in the        bottom (to keep a humid environment). Clean plain microscope        slides are placed at every second or third slide position in the        slide box to create rails or a platform onto which the        hybridization arrays can be placed. Each hybridization chamber        can hold two or three hybridization slides (depending on which        direction the slides are placed). The lid is carefully placed        onto the box and the box is then wrapped with plastic wrap.    -   Incubate on a level surface in a 37° C. incubator overnight        (about 16-18 hours)        Washing    -   Remove the cover slip by quickly but gently dipping the array in        1×SSC (let the cover slip slide off gently; hold the slide at        the bar-code end with forceps). Place the slide into a staining        rack and place into a staining dish (Evergreen Scientific        through Diamed cat#E/S258-4100-000) with fresh 1×SSC.    -   When all of the arrays have been removed from the hybridization        chambers, wash for 3 sets of 15 minutes each at 50° C. in clean        slide staining boxes containing pre-warmed (at 50° C.)        1×SSC/0.1% SDS with gentle occasional agitation    -   After the washes are complete, rinse the slides twice in room        temperature 1×SSC (plunging 4-6 times) and then in 0.1×SSC    -   Spin slides dry at 89×g for 5 minutes in a slide box lined with        Whatman paper. Alternatively, slides can be dried in a 50 mL        Falcon tube (and spun at 89×g for 5 min)    -   Arrays should be stored in the dark. It is recommended that        arrays be scanned as soon as possible after they are washed (at        least within two days). The hybridized slides of this invention        were scanned using Scanarray™ 4000 scanner (Perkin Elmer,        Wellesly, Mass.) at laser power setting varying between 65-75        and a PMT setting ranging from 50-55.

The TIFF images were quantified using ArrayVision v.8.0 (ImagingResearch Inc.). The data and images were then loaded into GeneTraffic™(Lobion Informatics) for normalization. The “Lowess, sub-grid” methodwas chosen for normalization in GeneTraffic™ Normalized intensity valueswere downloaded from the GeneTraffic™ database. The average S/N wascalculated in Excel for each slide type. The standard deviation for eachreplicate was calculated in Excel for each slide type. Each spot appearstwice on every array so even where only one array was tested there were2 replicates to calculate the standard deviation of the S/N betweenthem. Where more arrays were used the standard deviation of S/N wascalculated across all the replicate arrays and the replicate spots (2per array).

Signal to noise ratio was also measured for Ultragaps slides fromCorning Life Sciences. The procedure was identical to that mentionedabove except that less (80 μl) hybridization buffer was used. Also,scanning was performed at a different setting which was determined to beoptimum for these slides. Specifically, the laser power setting usedvaried between 95 to 100 and the PMT setting ranged from 70-80.

Precision Level

Precision level measurements were performed using arrays that werelabeled with the same sample of RNA in both the Cy5 and Cy3 channels.Ideally, all data points would fall exactly on a 45° line drawn throughthe origin on a scatter plot of normalized Cy3 signal intensity againstnormalized Cy5 signal intensity on a log-log plot for all the datapoints (M) on the slide. From this plot, the number of data points (N)that lie outside the Z-fold up and Z-fold down limits were determined.The Consistency or Specificity or Precision level is defined asZ-fold Precision Level, %=100×(1−(N/M))

Functionalized Substrate Examples

Sol-Gel Solution

A precursor solution was prepared by allowing 40.7 partstetraethoxysilane (Dynasil A made by Degussa Corporation, Parsippany,N.J.), 14.1 parts deionized water, 44.8 parts ethanol and 0.4 partshydrochloric acid (37%) to react for 24 hours at 65° C. The solution wasthen cooled and stored in a freezer until further use.

Silane Solution

A silane solution was prepared by mixing 2 parts ofaminopropyltriethoxysilane (A0750, United Chemical Technologies,Bristol, Pa.) to 98 parts of a 95/5 (w/w) mixture of ethanol/water. Thissolution was prepared just prior to use and was allowed to stand for atleast 5 minutes prior to its use.

Example 1

This example describes a highly functional microporous substrate of thepresent invention obtained by starting with ePTFE as the porousmaterial. The surprising advantages of the process and articles of thepresent invention are apparent when examining the substantialimprovement of the treated versus the untreated membranes.

Material Type: ePTFE

Membrane

An expanded polytetrafluoroethylene (ePTFE) membrane made in accordancewith the teachings of U.S. Pat. No. 4,187,390 was obtained. The ePTFEmembrane was about 74μ thick and had a bubble point of about 0.434 MPa(63 psi). Water beading on the surface of the membrane attested to itshydrophobicity.

Sol-Gel Treatment

The ePTFE membrane was treated by mounting the membrane on embroideryhoops for ease of handling and then immersing the membrane in a solutionobtained by diluting the sol-gel solution with equal amounts of ethanolby weight. After 5 minutes, the membrane was removed and immersed indeionized water for 5 minutes. Following the rinse step, the membranewas air dried and then heated at 150° C. for 5 minutes. At this stagethe membrane was hydrophilic and water readily wet the membrane.

Aminosilane Treatment

The hydrophilic membrane was further treated with aminosilane to providefunctional amino groups on its microstructure. This was accomplished byimmersing the membrane in the silane solution for 5 minutes, thenrinsing it in isopropyl alcohol (IPA) for 2 minutes and heating themembrane at 110° C. for 10 minutes. The membrane was silane treatedagain by repeating the identical steps of 5 minute immersion in silanesolution, 2 minute rinse in IPA and heating at 110° C. for 10 minutes.

Final Membrane

The resulting functionalized ePTFE membrane was 34 μm thick and aninhydrin assay indicated the amine density to be 416.4 nanomoles/cm²(or 121435 nanomoles/cm³). The auto fluorescence of the functionalizedePTFE membrane was measured as 21.2 and 30.4 respectively at 635 nm and532 nm. For comparison purposes, an identical but untreated ePTFEmembrane was tested for the presence of any functional amino group usingthe ninhydrin assay; no amino groups were detected.

Example 2

This example also describes a highly functional microporous substrate ofthe present invention obtained by starting with ePTFE as the porousmaterial. Example 1 was repeated except that PVOH was substituted forSol-gel. Again, the article of the present invention performed muchbetter than the untreated membrane of the same type as described inExample 1.

Material Type: ePTFE

PVOH Treatment

The ePTFE membrane used in Example 1 was treated by mounting themembrane in an embroidery hoop for ease of handling and then immersingthe membrane in IPA for 5 minutes, then in deionized water for 2minutes, then in 5 wt. % aqueous solution of polyvinyl alcohol (P1180,Spectrum Chemicals, Gardena, Calif.) for 10 minutes. The membrane wasremoved and immersed in deionized water for 10 minutes. Following therinse step, the membrane was air dried overnight under ambientconditions and then heated at 110° C. for 10 minutes. At this stage themembrane was hydrophilic and water readily wet the membrane.

Aminosilane Treatment

The hydrophilic membrane was further treated with aminosilane to providefunctional amino groups on its microstructure. This was accomplished byimmersing the membrane in the silane solution for 5 minutes, thenrinsing in isopropyl alcohol (IPA) for 2 minutes and heating themembrane at 110° C. for 10 minutes. The membrane was again silanetreated by repeating the identical steps of 5 minute immersion in silanesolution, 2 minute rinse in IPA and heating at 110° C. for 10 minutes.

Final Membrane

The resulting membrane was tested using the ninhydrin assay and theaverage density of the functional amino groups was detected to be 118.5nanomoles/cm².

Example 3

This example describes a highly functional microporous substrate of thepresent invention obtained by starting with microporous nylon as theporous material. The surprising advantages of the process and articlesof the present invention are apparent when examining the substantialimprovement of the treated versus the untreated membranes.

Material Type: Microporous Nylon

Membrane

A commercial microporous nylon membrane with surface treatment (HybondN+) was obtained from Amersham Biosciences Corp., Piscataway, N.J.

The membrane was about 150 μm thick and had a bubble point of about 12.5psi. Ninhydrin assay indicated that the functional amino group densityof the membrane as obtained from the vendor was 9.7 nanomoles/cm² (or638 nanomoles/cm³).

Sol-Gel Treatment and Aminosilane Treatment

The membrane was functionalized using the steps outlined in Example 1.

Final Membrane

The resulting membrane was about 147 μm thick and possessed a functionalgroup density of 1093 nanomoles/cm² (74199 nanomoles/cm³). This markedincrease in functional amino group density was a consequence of themethod of the present invention.

Example 4

This example describes a highly functional microporous substrate of thepresent invention obtained by starting with a porous ultrahigh molecularweight polyethylene (UHMWPE) sheet as the porous material. Thesurprising advantages of the process and articles of the presentinvention are apparent when examining the substantial improvement of thetreated versus the untreated membranes.

Material Type: Porous UHMWPE

Membrane

A commercial porous UHMWPE sheet (Porex 9619) was obtained from PorexCorporation, Fairburn, Ga. The sheet was about 1524 μm thick and itsbubble point was determined to be about 0.009 MPa (1.3 psi).

Sol-Gel Treatment and Aminosilane Treatment

The porous sheet was functionalized using the steps outlined in Example1.

Final Membrane

The resulting sheet was about 1524 μm thick and the functional groupdensity was 426.9 nanomoles/cm² (2801 nanomoles/cm³). Theauto-fluorescence of the functionalized UHMWPE sheet was measured as31.1 and 107.2 RFU respectively at 635 nm and 532 nm. For comparisonpurposes, the commercially obtained porous UHMWPE sheet was tested forthe presence of any functional amino group using the ninhydrin assay.The assay indicated that the functional amino group density of themembrane was 1.1 nanomoles/cm² (or 7.0 nanomoles/cm³). The autofluorescence of the commercially available UHMWPE sheet was measured as23.9 and 48.7 RFU respectively at 635 nm and 532 nm. This markedincrease in functional amino group density without significantlyincreasing the auto fluorescence level was due to the method of thepresent invention.

Example 5

This example describes a highly functional microporous substrate of thepresent invention obtained by starting with microporous polypropylene asthe porous material. The surprising advantages of the process andarticles of the present invention are apparent when examining thesubstantial improvement of the treated versus the prior art microporouspolypropylene membranes.

Material Type: Microporous Polypropylene

Membrane

A commercial microporous polypropylene membrane (Polysep, 0.1μ, catalogno. M01WP320F5) was obtained from GE Osmonics Inc., Watertown, Mass. Themembrane was about 86μ thick and its bubble point was determined to beabout 0.135 MPa (19.6 psi).

Sol-Gel Treatment and Aminosilane Treatment

The membrane was functionalized using the steps outlined in Example 1.

Final Membrane

The resulting membrane was about 74 μm thick and the functional groupdensity was now 486.8 nanomoles/cm² (66087 nanomoles/cm³). Forcomparison purposes, the commercially obtained microporous polypropylenemembrane was tested for the presence of any functional amino group usingthe ninhydrin assay. The assay could not detect any functional aminogroups on this membrane.

Example 6

This example describes a highly functional microporous substrate of thepresent invention obtained by starting with porous PTFE as the porousmaterial. The surprising advantages of the process and articles of thepresent invention are apparent when examining the substantialimprovement of the treated versus the untreated membranes.

Material Type: Porous PTFE

Membrane

A commercial porous polytetrafluoroethylene (PTFE) membrane (MuporPM17Y) was obtained from Porex Corporation, Fairburn, Ga. The membranewas about 152 μm thick and its bubble point was determined to be about0.044 MPa (6.4 psi).

Sol-Gel Treatment and Aminosilane Treatment

The porous membrane was functionalized using the steps outlined inExample 1.

Final Membrane

The resulting membrane was about 152 μm thick and the functional groupdensity was now 78.6 nanomoles/cm² (5158 nanomoles/cm³). No ninhydrinassay was performed on the untreated membrane since no functional aminogroups were expected to be present.

Example 7

This example describes a highly functional microporous substrate of thepresent invention obtained by starting with microporouspolyvinylidenefluoride as the porous material. The surprising advantagesof the process and articles of the present invention are apparent whenexamining the substantial improvement of the treated versus theuntreated membranes.

Material Type: Microporous Polyvinylidenefluoride Membrane

Membrane

A commercial microporous polyvinylidenefluoride membrane (PVDF-PlusTransfer membrane, 0.22μ, catalog no. PV2HY320F5) was obtained from GEOsmonics Inc., Watertown, Mass. The membrane was about 152μ thick andits bubble point was determined to be about 0.135 MPa (19.6 psi).

Sol-Gel Treatment and Aminosilane Treatment

The membrane was functionalized using the steps outlined in Example 1.

Final Membrane

The resulting membrane was about 154 μm thick and the functional groupdensity was now 420.6 nanomoles/cm² (27146 nanomoles/cm³). Theauto-fluorescence of the functionalized PVDF membrane was measured as173.4 and 526.5 RFU respectively at 635 nm and 532 nm. No ninhydrinassay was performed on the untreated membrane since no functional aminogroups were expected to be present. The auto fluorescence of theuntreated PVDF membrane was measured as 36.6 and 58.2 RFU respectivelyat 635 nm and 532 nm.

Example 8

This example describes a highly functional microporous substrate of thepresent invention obtained by starting with porous glass as the porousmaterial. This example describes the use of porous glass as thesubstrate. The surprising advantages of the process and articles of thepresent invention are apparent when examining the substantialimprovement of the treated versus the untreated substrates.

Material Type: Porous Glass

Substrate

Fabricated 25.4 mm×76.2 mm×1 mm thick rectangular slides made from Vycor7930 porous glass were obtained from Advanced Glass & Ceramics, Holden,Mass.

Aminosilane Treatment

Following the teachings of prior art, the porous glass slide wasfunctionalized using just the silane treatment steps specified inExample 1.

Sol-Gel Treatment and Aminosilane Treatment

Another sample of the porous glass slide was also functionalized usingboth the sol-gel and aminosilane steps outlined in Example 1.

Final Substrates

The slide that was only silane treated showed an amino group density of1169.6 nanomoles/cm² (or 12118 nanomoles/cm³) and the auto fluorescencelevels to be 23.9 and 93.7 RFU respectively at 635 nm and 532 nm. Incomparison, the porous glass slide that was functionalized using themethod of the present invention as outlined in Example 1 (i.e., treatedwith both sol-gel and aminosilane) indicated functional amino groupdensity of 1311.7 nanomoles/cm² (or 13590 nanomoles/cm³). The autofluorescence of this slide was measured as 36.3 and 332 RFU respectivelyat 635 nm and 532 nm. The ninhydrin assay was not performed on theuntreated substrate since no functional amino groups were expected to bepresent. The auto-fluorescence of the untreated porous glass slide wasmeasured to be 23.1 and 39.4 RFU respectively at 635 nm and 532 nm.

Composite Microarray Substrate Examples Comparative Examples

Commercially available microarray slides were obtained and analyzed forfunctional amino group density using the ninhydrin assay and for autofluorescence levels. The slides from Corning, Telechem and ErieScientific are all non-porous glass slides with aminofunctionalsurfaces. In comparison, the Vivid® Microarray slide from Pall Corp. isa microporous nylon polymer membrane adhesively bonded to a glass slide.Results for these commercial microarray slides are summarized in Table1.

TABLE 1 Amino Group Density, 635 nm - 532 nm - Name Source nanomoles/cm²Avg. RFU Avg. RFU Ultragaps slide Corning Life 4.8 21 23.4 SciencesArray-It Telechem 0.9 21 26.5 Superamine 2 International slideAminofunctional Erie 1.0 — — slide Scientific Company Vivid Pall 6.533.5 176 Microarray slide Corporation

Example 9

An ePTFE membrane was bonded to a glass slide, then functionalized.

Plain pre-cleaned glass microscope slides (VWR, catalog no. 48300) weretreated with the silane solution described above by dipping the slidesin the solution for 5 minutes, then rinsing them in IPA for 2 minutesand then heating them at 110° C. for 10 minutes. The silane treatedslides were then bonded to an ePTFE membrane that was 74 μm thick havinga bubble point of about 0.434 MPa (63 psi) and having an average fibrillength of 1.2 μm. The bonding was performed by spraying a 40 wt. %solution of TRABOND FDA2 epoxy adhesive (Tar-Con Inc., Bedford, Mass.)in methyl ethyl ketene onto the silane-treated glass slides using anair-brush kit (McMaster-Carr, catalog no. 9546T13).

The adhesive treated slides were placed on top of the ePTFE membranethat was secured in an embroidery hoop. The adhesive was then cured for60 minutes in a forced air oven set at 110° C. Following curing, theexcess ePTFE membrane was trimmed off the glass slide using a razorblade. The resulting composite slide had a layer of ePTFE membraneattached to one its surfaces. The membrane surface was hydrophobic. Aninhydrin assay indicated that no functional amino groups were present.Auto fluorescence of the membrane surface was expected to be about 21and 22 RFU respectively at 635 nm and 532 nm.

The composite slides were then place in a slide rack (Wheaton, catalogno. 900403) and the slides were treated with sol-gel by immersing themin the sol-gel solution diluted with equal parts in weight of ethanol.After 5 minutes of immersion, the slides were removed and rinsed inde-ionized water for 5 minutes. The rinsed slides were air dried andthen heated at 150° C. for 5 minutes. At this stage, the ePTFE membranewas extremely hydrophilic as evidenced by the fact that it readily wetwith water. These sol-gel treated slides were further treated byimmersing them into the silane solution for 5 minutes, then rinsing inIPA for 2 minutes and then heating them in an oven set at 110° C. for 10minutes. At this stage, the ePTFE membrane surface of the slidepossessed amino functionality. A ninhydrin assay indicated thefunctional amino group density to be 338.6 nanomoles/cm². Theauto-fluorescence level of the membrane surface of the slide wasmeasured to be 22.7 and 191.2 RFU at 635 nm and 532 nm, respectively.

Comparing these results with those of the prior art presented in Table 1demonstrates that the present invention provides a microarray slide withsignificantly higher functional amino group density while maintainingthe auto fluorescence level comparable to porous polymer membrane basedcommercial products.

Example 10

Plain pre-cleaned glass microscope slides (VWR, catalog no. 48300) werewiped clean with acetone and then bonded to an ePTFE membrane that was74 μm thick having a bubble point of about 0.434 MPa (63 psi) and havingan average fibril length of 1.2 μm. The bonding was done by manuallyspraying a 40 wt. % solution of TRABOND FDA2 epoxy adhesive containing1.8% (on epoxy solids) of 3-glycidoxypropoyltrimethoxysilane (G6720,United Chemical Technologies, Bristol, Pa.) in methyl ethyl ketone onthe glass slides by using an air-brush kit (McMaster-Carr, catalog no.9546T13) and then placing the adhesive treated slides on top of theePTFE membrane mounted on an embroidery hoop and curing the adhesive inan air circulating oven at 80° C. for 18 hours. Following curing, theexcess ePTFE membrane was manually trimmed from the glass slide using arazor blade. The resulting composite slide had a layer of ePTFE membraneattached to one surface. The membrane surface was hydrophobic andninhydrin assay indicated that no functional amino groups are present.Auto fluorescence of the membrane surface is expected to be about 21 and22 RFU at 635 nm and 532 nm respectively.

The composite slides were then placed in a slide rack (Wheaton, catalogno. 900403) and the slides were treated with sol-gel solution byimmersing it in the sol-gel solution diluted with equal parts in weightof ethanol. After 5 minutes of immersion, the slides are removed andrinsed in de-ionized water for 5 minutes. The rinsed slides were airdried and then heated at 150° C. for 5 minutes. At this stage, the ePTFEmembrane is extremely hydrophilic and readily wets out with water. Theseslides were further treated by immersing the sol-gel treated slides intothe silane solution for 5 minutes, then rinsing in IPA for 2 minutes andthen heating them at 110° C. for 10 minutes. At this stage, theauto-fluorescence level of the ePTFE membrane surface on the slide wasmeasured to be 27.4 and 350.8 RFU at 635 nm and 532 nm, respectively.

Example 11

The composite microarray slide described in Example 10 was processed atUHN in December 2005 to determine the average signal to noise ratio. Forcomparison, Ultragaps microarray slides (Corning) were also processed atthat time. A similar comparison was attempted using Vivid microarrayslide (Pall Corp.) using the same protocol. However, it was not possibleto print the complete array on these slides using the contact printingmethod used here.

Ambient ozone level is well known to have a significant effect on thestability of the signal in the Cy5 channel. Samples of the prior artthat are measured in the summer months exhibit much lower signal tonoise ratio values than identical samples measured in colder months. Thepreviously described inventive (Example 9) and prior art samples of thisexample had also been tested in March of 2005.

The signal to noise data appear in Table 2.

TABLE 2 Average Signal Average Signal Number of to Noise Ratio, to Noiseratio, Slide Description Slides Used Cy5 Cy3 inventive sample 2 205.8116.1 tested December. UltraGaps 5 110.1 81.35 sample tested Decemberinventive sample 2 191.5 93.7 tested March UltraGaps 2 87.8 40.2 sampletested March

This data demonstrates the significantly higher signal to noise ratiovalues for the inventive articles when compared against prior artarticles tested in the same time frame. The data also indicate theseasonal influence on the performance of the slides. The Cy5 signalsfrom the inventive microarray substrate show much less seasonalinfluence than those substrates representing the prior art. Theinvention sample shows about a 7% variance, while the conventionalsample shows about a 25% variance. The inventive sample thus hasenhanced stability (defined as less than 20%, and preferably less than10%, variability under the above conditions).

The FIGS. 3(A) and 3(B) show the scatter plots for inventive and priorart (Ultragaps) samples of this example, respectively, that were testedin December 2005. Also shown in these figures are the 2-fold, 1.5 foldand 1.2 fold limits from which different precision levels werecalculated. The precision level values are summarized in Table 3.

TABLE 3 Number of P_(1.5), 1.5-fold P_(1.2), 1.2-fold Slide slides P₂,2-fold precision precision Description Tested precision level, % level,% level, % Inventive 2 100 99.99 99.92 sample prior art 5 99.88 98.8892.73 sample

This data indicates that the microarray slides of the present inventionyield significantly higher precision levels as fold limits are madesmaller. That is, as the fold limits are made tighter, the precisionlevel of the inventive article is seen to maintain its extremely highprecision level, whereas the precision of the prior art article is seento significantly decrease. Consequently, substrates of the presentinvention generate more useful data for a given microarray experimentcompared prior art substrates. Also, the higher precision level affordedby the inventive substrate leads to higher degree of confidence in themicroarray data, thereby requiring less test replication.

Example 12

Another composite microarray slide prepared as per the proceduredescribed in Example 9 was processed at UHN in March 2005 to determinethe average signal to noise ratio. In this case, a 7407 clone set forthe mouse genome was used as the probe. For comparison, Ultragapsmicroarray slides (Corning) were also processed simultaneously. Signalto noise ratios were determined and the results are summarized in Table4.

TABLE 4 Average Signal Average Signal Number of to Noise Ratio, to Noiseratio, Slide Description Slides Used Cy5 Cy3 Inventive sample 2 75.854.5 UltraGaps 2 34.5 34.1 sample

The inventive microarray exhibited far higher signal to noise ratio forboth wavelengths compared to microarrays of the prior art.

Scatter plots of normalized Cy5 and Cy3 signal intensity were alsocreated for inventive and prior art (Ultragaps) samples of this example.The 2-fold, 1.5 fold and 1.2 fold limits were calculated from thescatter plots. The precision level values are summarized in Table 5.

TABLE 5 Number of P_(1.5), 1.5-fold P_(1.2), 1.2-fold Slide slides P₂,2-fold precision precision Description Tested precision level, % level,% level, % Inventive 2 99.98 99.91 97.87 sample UltraGaps 2 99.47 96.0973.12 sample

These data indicate that the microarray slides of the present inventionyield significantly higher precision level as fold limits are madesmaller. That is, as the fold limits are made tighter, the precisionlevel of the inventive article is seen to maintain its extremely highprecision level, whereas the precision of the prior art article is seento significantly decrease. Consequently, substrates of the presentinvention generate more useful data for a given microarray experimentcompared prior art substrates. Also, the higher precision level affordedby the inventive substrate leads to higher degree of confidence in themicroarray data, thereby requiring less test replication.

Example 13

The composite microarray slide prepared as per the procedure describedin Example 9 was processed at UHN in June 2005 to determine the averagesignal to noise ratio. In this case, a 19008 clone set from human genomewas used as the probe. For comparison, Ultragaps microarray slides(Corning) were processed simultaneously. In order to study the signalstability, the same slides were scanned on sequential days. The signalto noise ratios were determined and the results are summarized in Table6.

TABLE 6 inventive Inventive Ultragaps Ultragaps sample sample samplesample Dye Type Cy5 Cy3 Cy5 Cy3 Number of 3 3 3 3 Slides Tested Average88.9 54.8 11.5 25.6 S/N Ratio on Day1 Average 58.3 38.1 1.5 20.6 S/NRatio on Day2 Average 53.3 30.7 1.05 20.9 S/N Ratio on Day3

The data demonstrate that the microarray substrate of the presentinvention is significantly more effective in retaining the Cy5 signalover a longer time period.

The invention claimed is:
 1. An expanded polytetrafluoroethylenesubstrate comprising a microporous microstructure, said microporousmicrostructure having a fibril length between 0.5 and 5 microns, saidmicroporous microstructure ranging in thickness of 250 μm or less, aninterlayer over at least a portion of said microstructure, and afunctional layer attached to said interlayer, said functional layerhaving functional sites with a density of at least 50 nanomoles/cm². 2.A substrate as defined in claim 1 further comprising a functionalizedsite density between 2500 and 150,000 nanomoles/cm³.
 3. A substrate ofclaim 1 wherein said interlayer comprises polyvinylalcohol.
 4. Asubstrate of claim 1 wherein said functional sites comprise hydroxylgroups.
 5. A substrate of claim 1 wherein said functional sites compriseamine groups.
 6. A substrate of claim 1 wherein said functional sitescomprise carboxyl groups.
 7. A substrate of claim 1 wherein saidfunctional sites comprise aldehyde groups.
 8. A substrate of claim 1wherein said functional sites comprise epoxide groups.
 9. A substrate ofclaim 1 wherein said functional sites comprise nucleic acids.
 10. Asubstrate of claim 1 wherein said functional sites comprise proteins.11. A substrate of claim 1 wherein said functional sites comprisepeptides.
 12. A substrate of claim 1 wherein said functional sitescomprise oligonucleotides.
 13. A substrate of claim 1 wherein saidfunctional sites comprise antibodies.
 14. A substrate of claim 1 whereinsaid functional sites comprise cells.
 15. A substrate of claim 1 whereinsaid functional sites comprise enzymes.
 16. A substrate of claim 1wherein said functional sites comprise pathogens.
 17. A substrate ofclaim 1 used as a component of an active filter.
 18. A substrate ofclaim 1 used as a component of a blotting surface.
 19. A substrate ofclaim 1 used as a component of a diagnostic device.
 20. A substrate asdefined in claim 1 wherein said functional sites have a density of atleast 100 nanomoles/cm².
 21. A substrate as defined in claim 1 whereinsaid functional sites have a density of at least 250 nanomoles/cm². 22.A substrate as defined in claim 1 wherein said functional sites have adensity of at least 500 nanomoles/cm².
 23. A substrate as defined inclaim 1 wherein said functional sites have a density of at least 1000nanomoles/cm².
 24. A substrate of claim 1 wherein said interlayercomprises sol-gel coating.
 25. A substrate of claim 1 wherein saidfunctional layer comprises organosilane.
 26. A substrate of claim 1wherein said interlayer comprises sol-gel coating, and said functionallayer comprises organosilane.
 27. A substrate of claim 1 furthercomprising biomolecules bound to the functional sites.
 28. A method ofcreating a functionalized article comprising the steps of (1) providingan expanded polytetrafluoroethylene microporous substrate having amicrostructure, said microporous microstructure having a fibril lengthbetween 0.5 and 5 microns (2) depositing an interlayer over saidmicrostructure, (3) and attaching a functional layer to said interlayer,such that said article has a functional site density of at least 50nanomoles/cm², an interlayer over at least a portion of saidmicrostructure, and a functional layer attached to said interlayer, saidfunctional layer having functional sites with a density of at least 1000nanomoles/cm².
 29. A method as defined in claim 28 further comprising afunctional site density of between 2,500 and 150,000 nanomoles/cm³.